PROCESS FOR THE FERMENTATION OF FUNGAL STRAINS

Abstract

The present invention relates to a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side chains β-1,6-glycosidically bonded thereto, in a cascade of tanks using high-shear mixers.

Claims

1.-16. (canceled)

17. A process for fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto, in a cascade of tanks comprising at least a first tank (K1, K31) with a first volume (VK1, VK 31) and a second tank (K2, K32) with a second volume (VK2, VK32), comprising: a) fermenting the fungal strains in a first aqueous medium (M1, M31) in the first tank (K1, K31) and the volume of the first aqueous medium (VM1, VM31), resulting in a first mixture (S1, S31), b) transferring the first mixture (S1, S31) to the second tank (K2, K32), and c) fermenting the fungal strains in the first mixture (S1, S31) in a second aqueous medium (M2, M32) in the second tank (K2, K32) and the volume of the second aqueous medium (VM2, VM32), resulting in a second mixture (S2, S32), where the proportion of the volume of the first mixture (VM1, VM31) to the volume of the second tank (VK2, VK32) is in the range between ≧0.1% to ≦50% and where the first mixture (S1, S31) in step b) is passed through at least one high-shear mixer, the high-shear mixer (1) has a shearing geometry, such that the entire first mixture (S1, S31) entirely passes through the shearing geometry of the at least one high-shear mixer.

18. The process according to claim 17, wherein the high-shear mixer (1) is a rotor-stator mixer having a rotor (10) and a stator (20).

19. The process according to claim 18, wherein the rotor-stator mixer is a toothed-rim dispersing machine.

20. The process according to claim 18, wherein at least one of the rotor (10) and the stator of the rotor-stator mixer has at least two concentric toothed-rims (11, 12) and the other of the rotor and the stator (20) has at least one toothed rim (21, 22), wherein the at least one toothed-rim of the other of the rotor and the stator concentrically interleaves with the at least two concentric toothed-rims, wherein the first aqueous medium (M1, M31) passes through the interleaved toothed-rims.

21. The process according to claim 20, wherein the at least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed rim (21, 22) of the other of the rotor and the stator (20) have an equidistant tooth geometry and wherein the distance between adjacent teeth (13) of the respective outer toothed-rim (11) is larger than the distance between adjacent teeth (23) of the respective inner toothed-rim (21), wherein the first aqueous medium M1 passes through the interleaved toothed-rims in a direction of ascending teeth distance.

22. The process according to claim 20, wherein the first mixture (S1) passes through a gap (2) in radial direction, which gap in a radial direction is formed by the concentrically interleaving at least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20), wherein the gap (2) between an outer diameter of a toothed rim and an inner diameter of a radial outwardly adjacent toothed-rim has a width between 0.2 mm and 2.0 mm.

23. The process according to claim 20, wherein the first mixture (S1) dwells for between 0.01 s and 0.004 s while passing the least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20).

24. The process according to claim 19, wherein edges (14, 24) of teeth (13, 23) along a flow path through the shearing geometry have rounded edges with a radius of at least 0.2 mm.

25. The process according to claim 18, wherein the rotor (10) rotates at a speed relative to the stator between 250 and 7200 revolutions per minute.

26. The process according to claim 18, wherein the rotor (10) rotates at a peripheral speed between 2 m/s and 60 m/s.

27. The process according to claim 17, wherein the proportion of the volume of the first mixture (VM1, VM31) to the volume of the second tank (VK2, VK32) is in the range between ≧1% to ≦20%.

28. The process according to claim 17, wherein the at least one beta-glucan is selected from the group consisting of Schizophyllan and Scleroglucan, wherein the Schizophyllan or Scleroglucan are obtained by fermentation of fungal strains.

29. The process according to claim 17, wherein the fungal strains are Schizophyllum commune or Sclerotium rolfsii.

30. A process according to claim 17, wherein the tank cascade further comprises a third tank (K33) with a third volume (VK33), and the process for fermentation further comprises: d) transferring the second mixture (S32) to the third tank (K33), and e) fermenting the fungal strains in the second mixture (S32) in a third aqueous medium (M33) in the third tank (K33), wherein the proportion of the second mixture to the volume of the third tank (VK33) is in the range between ≧0.1% to ≦50%.

31. The process according to claim 30 wherein the second mixture (S32) in step d) is passed through at least one high-shear mixer, the high-shear mixer (1) has a shearing geometry, such that the entire second mixture (S32) entirely passes through the shearing geometry of the at least one high-shear mixer.

32. The process according to claim 30, wherein the proportion of the second mixture (S32) to the volume of the third tank (VK33) is in the range between ≧1% to ≦20%.

33. The process according to claim 20, wherein the first mixture (S1) passes through a gap (2) in radial direction, which gap in a radial direction is formed by the concentrically interleaving at least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20), wherein the gap (2) between an outer diameter of a toothed rim and an inner diameter of a radial outwardly adjacent toothed-rim has a width between 0.4 mm and 1.2 mm.

34. The process according to claim 20, wherein the first mixture (S1) passes through a gap (2) in radial direction, which gap in a radial direction is formed by the concentrically interleaving at least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20), wherein the gap (2) between an outer diameter of a toothed rim and an inner diameter of a radial outwardly adjacent toothed-rim has a width between 0.8 mm and 0.9 mm.

35. The process according to claim 20, wherein the first mixture (S1) dwells for between 0.02 and 0.07 s while passing the least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20).

36. The process according to claim 20, wherein the first mixture (S1) dwells for 0.01 s+/−0.001 s while passing the least two concentric toothed-rims (11, 12) of one of the rotor (10) and the stator and the at least one toothed-rim (21, 22) of the other of the rotor and the stator (20).

37. The process according to claim 19, wherein edges (14, 24) of teeth (13, 23) along a flow path through the shearing geometry have rounded edges with a radius of more than 3 mm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Exemplary embodiments of the present invention will be described in the following with reference to the following drawings.

[0092] FIG. 1. illustrates a two-step fermentation process with an interleaved shearing process according to an exemplary embodiment.

[0093] FIG. 2. illustrates a three-step fermentation process with two interleaved shearing processes according to an exemplary embodiment.

[0094] FIG. 3. illustrates a four-step fermentation process with three interleaved shearing processes according to an exemplary embodiment.

[0095] FIG. 4. illustrates a cross sectional view of a high shear mixing geometry according to an exemplary embodiment.

[0096] FIG. 5a. illustrates a top view of one of a rotor and a stator of a high shear mixer according to an exemplary embodiment.

[0097] FIG. 5b. illustrates a top view of the other of a rotor and a stator of a high shear mixer according to an exemplary embodiment in view of FIG. 5a.

[0098] FIG. 6. illustrates a detailed cut out of a cross sectional view of a high shear mixer geometry according to an exemplary embodiment.

[0099] FIG. 7 illustrates an exemplary space-time-yield over time chart for a laboratory fermenter with/without morphology control.

[0100] FIG. 8 illustrates an exemplary space-time-yield over time chart for a pilot plant fermenter with/without morphology control.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0101] FIG. 1 illustrates a two-step fermentation process with an interleaved shearing process according to an exemplary embodiment. FIG. 1 in particular illustrates the general set-up of the tank and shear mixer structure. A first tank K1 having a first tank volume VK1 and receives a first aqueous medium M1. A fermentation of fungal strains takes place in the first aqueous medium M1, resulting in a first mixture S1. During fermentation, the fungal strains form agglomerates. The first mixture S1 including the agglomerates of fungal strains is transferred to a second tank K2 having a second tank volume VK2. A second aqueous medium M2 may be added to the first mixture S1, so that a further fermentation of fungal strains in the first mixture in a second aqueous medium in the second tank takes place, resulting in a second mixture S2. As the agglomerates in the first mixture before being transferred from the first tank K1 to the second tank K2 are large and do not allow an efficient fermentation process in the second tank, the first mixture S1 flows through a high-shear mixer 1 being arranged between the first tank K1 and the second tank K2. The proportion of the volume of the first mixture VM1 to the volume of the second tank VK2 is in the range between 0.1% to 50%. The high-shear mixer 1 is of a type in view of a shearing geometry, such that the entire first mixture S1 entirely passes through the shearing geometry of the high-shear mixer 1. The detailed geometry of the high-shear mixer is later described with respect to FIGS. 4, 5a, 5b and 6.

[0102] FIG. 2 illustrates a three-step fermentation process with two interleaved shearing processes according to an exemplary embodiment. FIG. 2 illustrates a first tank K31 with a first tank volume VK31. A first aqueous medium M31 is in the first tank volume VK31. A fermentation of fungal strains takes place in the first aqueous medium M31 in the first tank volume VK31, resulting in a first mixture S31. The first mixture S31 is transferred to the second tank K32 having a second tank volume VK32. An aqueous medium M32 is added to the first mixture S31 in the second tank volume VK32, so that a fermentation of fungal strains in the first mixture in the second aqueous medium M32 takes place. As the fungal strains form agglomerates during fermentation in the first tank, the size of the agglomerates should be reduced, e.g. by a shearing process by a high-shear mixer 1 being arranged between the first tank K31 and the second tank K32. Consequently, the first mixture S31 flows through the high-shear mixer 1 and will be sheared, and then enters the second tank K32. The proportion of the volume of the first mixture VM31 to the volume of the second tank K32 may be in the range between 0.1% to 50%. The first mixture S31 entirely passes through the high-shear mixer 1, wherein the high-shear mixer 1 has a shearing geometry, such that the entire first mixture S31 entirely passes through the shearing geometry of the high-shear mixer 1. This means that the shear mixer has a flow through geometry. After a further fermentation of fungal strains in the first mixture and the second aqueous medium M32, the resulting second mixture S32 will be transferred to a third tank K33. The second mixture S32 for this purpose passes a further high-shear mixer 1 so that the again formed agglomerates will be again sheared before entering the third tank K33. In the third tank, the second mixture S32 will be added to a third aqueous medium M33, so that a further fermentation can take place in the volume VK33 of the third tank K33.

[0103] FIG. 3 illustrates a four-step fermentation process with three interleaved shearing processes according to an exemplary embodiment. Fungal strains in a first aqueous medium M41 in a volume VK41 in a first tank K41 are fermented, resulting in a first mixture S41. During fermentation process, fungal strains form agglomerates, which possibly do not allow an efficient further fermentation, so that the agglomerates should be sheared before starting a further fermentation in the second tank K42 having a second tank volume VK42. Thus, the first mixture S41 is transferred to the second tank K42 and during transfer passes the high-shear mixer 1 between the first tank K41 and the second tank K42. The first mixture S41 including the sheared agglomerates will be added to a second aqueous medium M42, so that a further fermentation may take place, resulting in a second mixture S42. The second mixture S42 will then be transferred to a third tank K43 having a third tank volume VK43. The second mixture S42 passes a high-shear mixer 1, so that the agglomerates being formed during the second fermentation will be sheared. The second mixture in the third tank K43 will be added to a third aqueous medium M43. Thus, a third fermentation process may take place in the tank volume VK43, resulting in a third mixture S43. Also the third mixture S43 may include agglomerates which may decrease efficiency of a further fermentation. Therefore, the third mixture S43 also passes a high-shear mixer 1 before entering a fourth tank K44 having a fourth tank volume VK44. In the fourth tank volume VK44, the third mixture S43 will be added to a fourth aqueous medium M44. A further fermentation may take place in the fourth tank volume VK44.

[0104] It should be noted, although not explicitly described, that also a fermentation process can be provided having more than four steps as described above with respect to FIG. 3. It should be noted that the high-shear mixers 1 between two respective tanks may have different specifications according to the expected structure of the agglomerates in the respective tank after fermentation.

[0105] Further, it should be noted that in all three embodiments as described above FIGS. 1, 2 and 3, the proportion of the volume of the first mixture VM1, VM31, VM41, to the volume of the second tank VK2, VK32, VK42 may be in the range between 0.1% and 50%. Further, it should be noted, that for all three above described embodiments with respect to FIGS. 1, 2 and 3, the proportion of the volume of the first mixture VM1, VM31, VM41, to the volume of the second tank VK2, VK32, VK42 may be in a range between ≧1% and 20%.

[0106] Further, it should be noted that for the embodiments described with respect to FIGS. 2 and 3, i.e. the three-step fermentation process and the four-step fermentation process, the proportion of the second mixture S32, S42 to the volume of the third tank VK33, VK43 may be in a range between 0.1% and 50%, and in particular between ≧1% and 20%.

[0107] Additionally, the proportion of the third mixture S43 to the volume of the fourth tank VK44 in the embodiment described with respect to FIG. 3 may be in a range between 0.1% to 50%, and in particular between ≧1% and 20%.

[0108] FIG. 4 illustrates a cross-sectional view of a high-shear mixing geometry according to an embodiment. A high-shear mixer according to the illustrated embodiment of FIG. 4 comprises a rotor 10 and a stator 20. The rotor has a first toothed-rim 11 having a plurality of teeth 13. The rotor 10 further has a second toothed-rim 12 also comprising a plurality of teeth 13. The stator 20 also has a first toothed-rim 21 having a plurality of teeth 23. Further, the stator has a second toothed-rim 22 also having a plurality of teeth 23. The teeth of each of the toothed-rims 11, 12, 21, 22 are arranged along a circuit being concentric to the rotational axis of the high-shear mixer 1. The toothed-rims of the rotor 11, 12 and the toothed-rims of the stator 21, 22 interleave so as to form a gap 2 between the teeth as such, and the rotor and stator body, respectively. The mixture to be sheared will be fed through for example a through-hole of the rotor 10 and flows along the double arrows in FIG. 4, so that the mixture S1 will be sheared between teeth of adjacent rims. It should be noted, that the feeding of the mixture S1 can also take place through a through-hole of the stator, although this specification is not explicitly illustrated in FIG. 4. Further, it should be noted that the number of toothed-rims of the rotor as well as the stator may be more than two.

[0109] FIG. 5a illustrates a top view of one of a rotor and a stator of a high-shear mixer according to an embodiment. In particular, FIG. 5a illustrates a rotor 10 having a first toothed-rim 11 including a plurality of teeth 13. Further, a second toothed-rim 12 is provided on the rotor. It should be noted, that the configuration illustrated in FIG. 5a may also be a configuration for a stator. The teeth 13 of the first and second toothed-rims 11, 12 may be different as well as the width of the teeth and the width of the gap there between in a circumferential direction.

[0110] FIG. 5b illustrates a top view of the other of a rotor and a stator of a high-shear mixer according to an embodiment in view of FIG. 5a, and in particular a stator 20. The stator 20 has at least one rim 21 having a plurality of teeth 23. As can be seen by the dashed lines between FIG. 5a and FIG. 5b, the toothed-rims of the rotor 10 and the stator 20 interleave when being coupled as illustrated in FIG. 4.

[0111] FIG. 6 illustrates a detailed cut-out of a cross-sectional view of a high-shear mixer geometry according to an exemplary embodiment. FIG. 6 illustrates the rotor 10 and the stator 20 with respective teeth of a toothed-rim. It should be noted that the rotor 10 and/or the stator 20 may have a further toothed-rim with a similar geometry. The teeth 13 and 23 of the toothed-rims 11 and 21 of the rotor 10 and the stator 20, respectively have rounded edges. The edges have a radius R so as to provide a smooth transition between the teeth and the stator body or the teeth and the rotor body, as well as between the teeth and the gap 2. The rounded edges 14, 24 result in a reduced impact to the agglomerates of the mixture, so that the agglomerates are not cut or destroyed by sharp edges of the teeth 13, 23, which will result in a deteriorated fermentation process. It should be noted that rounded edges may be provided in particular at edges between teeth of adjacent rims. Further, rounded edges can also be provided between adjacent teeth of a single rim. The radius R of teeth of adjacent rims may be adapted to each other so as to have a more or less continuous width of the gap 2.

Examples

[0112] The Schizophyllum commune strain used is laid open in EP 0 504 673.

[0113] Suitable nutrient media for the precultures and main cultures and cultivation conditions can be found for example in the patent EP 504 6073, EP 0 271 907 and “Process and molecular data of branched 1,3-β-D-glucans in comparison with Xanthan, U. Rau, R.-J. Müller, K. Cordes, J. Klein, Bioprocess Engineering, 1990, Volume 5, Issue 2, pp 89-93” and “Udo Rau, “Biosynthese, Produktion und Eigenschaften von extrazellularen Pilz-Glucanen [Biosynthesis, production and properties of extracellular fungal glucans]”, Postdoctoral thesis, Technical University of Braunschweig, 1997″.

[0114] Nutrient medium used: 30 g/l glucose, 3 g/l yeast extract, 1 g/l KH.sub.2PO.sub.4, 0.5 MgSO.sub.4*.sub.7 H.sub.2O

1. Preculture

[0115] Strain maintenance and cultivation of the biomass are described for example in “Oxygen controlled batch cultivations of Schizophyllum commune for enhanced production of branched β-1,3-glucans, U. Rau, C. Brandt Bioprocess Engineering September 1994, Volume 11, Issue 4, pp 161-165”. The ratio of the volumes upon transfer was about 5%.

[0116] All of the tanks of the preculture were operated at a constant speed and gassing rate so that the pO.sub.2 was always above 60%. The duration of the precultures was chosen such that the glucose did not drop below 5 g/l.

2. Main Culture

[0117] The main culture was carried out according to the process described in the literature under oxygen-limiting conditions. The procedure for the main culture is described for example in “Oxygen controlled batch cultivations of Schizophyllum commune for enhanced production of branched β-1,3-glucans, U. Rau, C. Brandt Bioprocess Engineering September 1994, Volume 11, Issue 4, pp 161-165”, “Udo Rau, “Biosynthese, Produktion und Eigenschaften von extrazellulären Pilz-Glucanen [Biosynthesis, production and properties of extracellular fungal glucans]”, Postdoctoral thesis, Technical University of Braunschweig, 1997” and “Process and molecular data of branched 1,3-β-D-glucans in comparison with Xanthan, U. Rau, R.-J. Müller, K. Cordes, J. Klein, Bioprocess Engineering, 1990, Volume 5, Issue 2, pp 89-93”,

3. Transfer of the Preculture to the Main Culture with Rotor-Stator Mixer

[0118] The increase in volumetric productivity in the main culture through the use of a toothed-wheel pump in the bypass, as described in DE 4012238 A1, could not be recreated. The opposite effect was observed in experiments that the recirculation via a bypass, as described in DE 4012238 A1, significantly reduces the volumetric productivity in the main culture.

[0119] Surprisingly, it was found that using a continuously operated rotor-stator mixer when transferring the preculture to the main culture leads to a significant increase in the STY. In this example, a rotor-stator mixer from Cavitron was used, bench instrument CD 1000 equipped with a chamber system, operated at 5-20 l/min, peripheral speed: 3-50 m/s.

[0120] The rotor-stator mixer was incorporated into the pipeline of the last tank of the preculture to the main culture tank in the reactor cascade and steam-sterilized prior to insertion in order to permit aseptic operation.

4. Determination of the Space-Time Yield

[0121] The space-time yield (STY), also called volumetric productivity, was determined by measuring the glucan concentration in a sample taken after a runtime of 72 h using a method described in the literature. The measured concentration divided by the runtime until the sample was taken (72 h) gives the space-time yield. For the purposes of simplification, relative STY are shown. The STY which were achieved without using a high-shear mixer were set as 100%.

5. Determination of the Filtration Ratio (FR Value)

[0122] Principle of Measurement:

[0123] In the determination of the filtration ratio (FR value), the amount of filtrate which runs through a defined filter is determined as a function of time. The FR value is determined according to the following formula (I)

FR=(t.sub.190g−t.sub.170g)/(t.sub.70g−t.sub.50g)  (I),

where the variables and the equation have the following meaning:
t.sub.190g=time in which 190 g of filtrate are obtained,
t.sub.170g time in which 170 g of filtrate are obtained,
t.sub.70g=time in which 70 g of filtrate are obtained,
t.sub.50g=time in which 50 g of filtrate are obtained.

[0124] Thus, in each case the time span which is required for in each case 20 g of filtrate to flow through is determined, i.e. at a early time and at a late time in the filtration process, and the quotient is calculated from the two time spans. The larger the FR value, the more greatly is the filtration velocity slowed down with increasing duration of the filtration process. This indicates increasing blockage of the filter, for example by gels or particles.

[0125] The FR value is determined by the following method:

5.1. Equipment

[0126] a) Sartorius pressure filtration apparatus 16249; filter diameter 47 mm; with 200 ml digestion cylinder (Øi=41 mm)
b) Isopore membrane 1.2 μm; Ø 47 mm; No. RTTP04700 available from Merck Millipore

c) Balance

5.2 Preparation of the Glucan Solution

[0127] First, 50 g of a mixture of the glucan solution obtained from the experiments and water is prepared, i.e. in a ratio such that the concentration of the glucan is 1.75 g/l. The mixture is stirred for 10 min and checked visually for homogeneity. If the mixture is still inhomogeneous, further stirring is effected until the mixture is homogeneous. The mixture is then made up to a total amount of 250 g with 200 g of ultrapure water. Thereafter, stirring is effected for at least 1 h for homogenization, after which the pH is adjusted to 6.0 with 0.1 M NaOH and stirring is then effected again for 15 min. The pH of 6.0 is checked again. The final concentration of the glucan in the mixture is 0.35 g/l.

5.3. Carrying Out the Filtration Test

[0128] The filtration test is effected at room temperature (T=25° C.) at a pressure of 1.0 bar (compressed air or N.sub.2). [0129] place coarse support grid on the sieve tray [0130] place fine support grid on the sieve tray [0131] place membrane filter on top [0132] insert seal (O-ring) [0133] screw sieve tray and outlet tap to the cylinder [0134] close outlet tap [0135] introduce 220 g (about 220 ml) of solution [0136] screw upper cover to cylinder [0137] clamp on inlet air tube [0138] check pressure and adjust to 1.0 bar [0139] place beaker on the balance under the filtration apparatus. Press tare. [0140] open outlet tap [0141] the test is stopped when no more filtrate emerges.

[0142] By means of the balance, the amount of filtrate is determined as a function of time. The mass indicated in each case can be read visually but of course also automatically and evaluated.

[0143] FIG. 7 illustrates an exemplary relative space-time-yield over time chart for a laboratory fermenter with/without morphology control. As can be seen from FIG. 7, a laboratory fermenter with morphology control has a higher relative space-time-yield compared to a laboratory fermenter without a morphology control. Thus, the efficiency of the laboratory fermenter with morphology control is higher than a laboratory fermenter without a morphology control. In particular, FIG. 7 shows the comparison of the relative STY for production on a laboratory scale (21 l) with a three-stage preculture. It can be seen that the STY is significantly increased in the case of morphology control to avoid pellet or agglomerate formation.

[0144] FIG. 8 illustrates an exemplary relative space-time-yield over time chart for a pilot plant fermenter with/without morphology control. As can be seen from FIG. 8, the relative space-time-yield of a pilot plant scale fermenter with morphology control is a little bit higher than a relative space-time-yield of a pilot plant scale fermenter without morphology control. In particular, FIG. 8 shows the comparison of the relative STY for the production on a pilot-plant scale (3 m.sup.3) with a three-stage preculture. It can be seen that the STY is significantly increased in the case of morphology control to avoid pellet/agglomerate formation.

REFERENCE LIST

[0145] 1 high shear mixer [0146] 2 gap [0147] 10 rotor [0148] 11 toothed rim of rotor [0149] 12 toothed rim of rotor [0150] 13 tooth/teeth of toothed rim of rotor [0151] 14 edge of tooth [0152] 20 stator [0153] 21 toothed rim of stator [0154] 22 toothed rim of stator [0155] 23 tooth/teeth of toothed rim of stator [0156] 24 edge of tooth [0157] K1 first tank [0158] K2 second tank [0159] K31 first tank [0160] K32 second tank [0161] K33 third tank [0162] K41 first tank [0163] K42 second tank [0164] K43 third tank [0165] K44 fourth tank [0166] M1 first aqueous medium [0167] M2 second aqueous medium [0168] M31 first aqueous medium [0169] M32 second aqueous medium [0170] M33 third aqueous medium [0171] M41 first aqueous medium [0172] M42 second aqueous medium [0173] M43 third aqueous medium [0174] M44 fourth aqueous medium [0175] S1 first substance [0176] S2 second substance [0177] S31 first mixture [0178] S32 second mixture [0179] S41 first mixture [0180] S42 second mixture [0181] S43 third mixture [0182] VK1 first tank volume [0183] VK2 second tank volume [0184] VK31 first tank volume [0185] VK32 second tank volume [0186] VK33 third tank volume [0187] VK41 first tank volume [0188] VK42 second tank volume [0189] VK43 third tank volume [0190] VK44 fourth tank volume [0191] VM1 volume of first aqueous medium [0192] VM2 volume of second aqueous medium [0193] VM31 volume of first aqueous medium [0194] VM32 volume of second aqueous medium [0195] VM33 volume of third aqueous medium [0196] VM41 volume of first aqueous medium [0197] VM42 volume of second aqueous medium [0198] VM43 volume of third aqueous medium [0199] VM44 volume of fourth aqueous medium